The overall goal of this research project is to identify genes which are responsible for the in vitro conversion of normal human oral keratinocytes to tumorigenic cells, and in doing so to test the hypothesis that human oral cancer arises by sequential activation or inactivation of multiple genes, e.g., growth factors, growth factor receptors, tumor suppressor genes, and oncogenes. We have previously established an in vitro oral carcinogenesis model; We immortalized normal human oral keratinocytes by transfecting them with cloned "high risk" human papillomavirus (HPV) DNA such as HPV-16 and HPV-18 DNA, and subsequently converted the immortalized cells to tumorigenic cells by exposing them to tobacco-related chemical carcinogens. When normal cells were exposed to chemical carcinogens they failed to convert to cancer cells and always underwent to apoptosis. Though the immortalization of normal human oral keratinocytes is known to be induced by viral oncoproteins such as E6 and E7 which inactivate p53 and Rb tumor suppressors, the mechanisms of the chemical carcinogen-induced conversion of the immortalized cells to tumorigenic cells remain unknown. In the present proposal we plan to search for already identified or unidentified genes which may be responsible for the conversion of the immortalized cells to tumor cells by conducting mRNA differential display, cloning of cDNAs which differently expressed from the immortalized and tumor cells, constructing expression vectors that express cloned cDNAs, and determining the alterations in the phenotypes of cells transfected with the vectors. The full-length of at least 6 unidentified (unknown) genes will be cloned and their biological activities on the cell proliferation, on the in vitro anchorage independency on the in vivo tumorigenicity, and on the differentiation potential of cells will be tested in the immortalized and tumorigenic cells. Similar studies will be performed by using known genes'cDNAs whose expression patterns in the above two cell lines are clearly different.